ABSTRACT Progressive Multifocal Leukoencephalopathy (PML), a life-threatening demyelinating brain disease caused by JC polyomavirus (JCV), is a significant complication for patients with HIV/AIDS, hematologic malignancies, rheumatologic diseases, and those receiving long-term immunomodulating therapies. The immunomodulating therapy mostly closely associated with PML is natalizumab, an ?4 integrin antibody (Ab) commonly used to treat multiple sclerosis (MS). Lack of a tractable animal model for PML is a widely recognized hurdle to defining the mechanisms of immunological control of JCV CNS infection. Using mouse polyomavirus (MuPyV), our laboratory developed a robust model of polyomavirus-associated demyelinating leukoencephalitis, with viral infection and T cell infiltration. This proposal seeks to use the MuPyV-CNS infection model to mechanistically understand the cellular and humoral immune mechanisms in place to control JCV CNS infection and how immunosuppression predisposes patients to JCV CNS infection. Increasing evidence supports the importance of CD4+ T cells in controlling JCV and thereby preventing PML. Recent studies in our laboratory show that CD4+ T cells are essential for differentiation and maintenance of MuPyV-specific tissue-resident memory CD8+ T cells (TRMs) in the brain. My preliminary data strongly supports the likelihood that IL-21 is a major component of the help provided by CD4+ T cells. I have further found that the serum from IL-21 receptor (IL21R)-deficient mice has a reduced ability to neutralize MuPyV carrying an amino acid mutation in its VP1 capsid protein that corresponds to a frequent VP1 mutation in PML-JCV. In Specific Aim 1, I hypothesize that IL-21 is required for brain-infiltrating CD8+ T cells to differentiate into TRMs. To test this hypothesis, I will define a time course for IL-21 production in the MuPyV-infected brain, its cellular sources, and when its receptor is expressed by MuPyV-specific CD8+ T cells. Using donor MuPyV-specific CD8+ T cells lacking IL21R, I will determine if signaling through the IL21R is intrinsically required by CD8+ T cells for their differentiation into TRMs. I will also apply RNA-seq to determine if the transcriptomes of IL21R-/- CD8+ T cells in the brain are similar to CD8+ T cells in CD4+ T cell-deficient mice. In Specific Aim 2, I hypothesize that IL21R signaling diversifies the VP1 Ab repertoire in a B cell-intrinsic manner to facilitate recognition of a MuPyV carrying a PML-synonymous VP1- mutation. To test this hypothesis, I will use MuPyV Ab neutralization assays, ELISAs, and germinal center immunohistochemistry to define the time course for developing a hole in the VP1 Ab specificity repertoire under T-independent (TCR?-/-) and IL-21-deficient (IL21R-/-) conditions. I also will determine plasma cell specificity against the PML-synonymous VP1 MuPyV mutant using fluorescently labeled MuPyVs in these aforementioned conditions. Finally, I will distinguish whether the mechanistic basis for the diminished VP1 neutralizing Ab repertoire is due to an intrinsic defect in IL21R signaling in B and/or T cells.